CN110305055A - A kind of fluorescence probe and the preparation method and application thereof identifying mitochondria viscosity - Google Patents
A kind of fluorescence probe and the preparation method and application thereof identifying mitochondria viscosity Download PDFInfo
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- CN110305055A CN110305055A CN201910626990.2A CN201910626990A CN110305055A CN 110305055 A CN110305055 A CN 110305055A CN 201910626990 A CN201910626990 A CN 201910626990A CN 110305055 A CN110305055 A CN 110305055A
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- viscosity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
Abstract
The present invention relates to a kind of fluorescence probe and the preparation method and application thereof for identifying mitochondria viscosity more particularly to a kind of fluorescence probe based on Benzazole compounds identification mitochondria viscosity in vivo with external research；Belong to small organic molecule fluorescence probe field.The probe is EIMV, and chemical structural formula is as shown in the formula (I).The present invention also provides the preparation methods of the probe, and disclose the fluorescence probe spectral properties such as change in fluorescence and selectivity in the detection process.Experiments have shown that: it is provided by the invention to identify mitochondria and there is the fluorescence probe for being able to detect viscosity, and can be realized intracellular imaging, it studies for biology as indicating that it has potential application in fluorescent biolabels field using theoretical basis has been established.(I).
Description
Technical field
The present invention relates to a kind of fluorescence probes and the preparation method and application thereof for identifying mitochondria viscosity, more particularly to one kind
Based on Benzazole compounds identification mitochondria viscosity fluorescence probe in vivo with external research；Belong to small organic molecule fluorescence
Probe field.
Background technique
Mitochondrial matrix viscosity is closely related by changing mitochondria network structure and mitochondrial respiratory state, passes through line grain
The diffusion of the adjustable metabolite of change of body matrix viscosity.Mitochondria correlation viscosity can lead to diabetes extremely, nerve moves back
Row disease and cancer.Therefore, effective technology is developed to monitor the variation of mitochondria viscosity and be very important.
Currently, traditional mechanical measuring means has very much, such as drop ball viscometer, capillary viscometer.However, these
Method is all only applicable in traditional liquid and is not suitable for living cells, it is therefore desirable to develop new tool to detect intracellular viscosity.
Fluorescence probe comes due to having the advantages that highly sensitive, highly selective and real time imagery as a kind of powerful tool
Detect the organelle viscosity of living cells.Molecule rotor is non-emissive wave in low-viscosity media, is had in resisting medium strong
Fluorescence provides a kind of useful method to detect intracellular viscosity.Most of probes have nonspecific fixed in cell
Position is not suitable for the microviscosity for detecting certain organs device.Up to the present, although there are many reports of mitochondria viscosity probe
Road, but distinguish normal cell and tumour cell and the case where further discriminating between normal organ and diseased organ is rarely found.Therefore,
The Mitochondrially targeted probe of viscosity sensitivity is still challenging to the mitochondria with anti-interference ability.
Summary of the invention
In view of the deficiencies of the prior art, the present invention has synthesized Mitochondrially targeted viscosity fluorescence probe by MOLECULE DESIGN,
With the increase of viscosity, the fluorescence intensity of probe is significantly increased, the sensitivity with height.And it can be to intracellular microenvironment
Viscosity carry out fluorescence imaging.
A kind of fluorescence probe identifying mitochondria viscosity, is named as EIMV, chemical structural formula is as shown in the formula (I):
()
The preparation method of above-mentioned fluorescence probe, comprising the following steps:
Compound 1 and pyridiniujm 2 are made into solvent with methanol, reacts, is heated to reflux under nitrogen protection；Column chromatography obtains chemical combination
Object EIMV.
Application of the above-mentioned fluorescence probe in terms of spectrum and cell.
Above-mentioned fluorescence probe realizes the identification to mitochondria viscosity in a manner of fluorescence enhancement, viscous applied to cell mitochondrial
The measurement of degree.
Above-mentioned fluorescence probe can also be applied to living cells mitochondria viscosity measurement.
Fluorescence probe EIMV fluorescence in pure aquatic system of the present invention is weaker, and in tacky glycerin solution
Fluorescence enhancement, it was demonstrated that the probe may be viscosity probe (Fig. 2).
It in viscosity Choice tests, is excited when with 510nm light, 86 times of fluorescence only occurs when viscosity enhances and increases
By force.(Fig. 3)；As buffer PBS and glycerine system kind glycerol content are increased, (Fig. 4) is remarkably reinforced in fluorescence intensity.
When being imaged in the cell, the mitochondrial probe of probe and business carries out common location experiment it can be found that common location
Coefficient can achieve 0.90(Fig. 5)；When excitation wavelength is 560nm, probe has faint fluorescence in the cell, when with can not bacterium
(Fig. 6) is remarkably reinforced in fluorescence after element and nystatin processing.
Beneficial effects of the present invention:
(1) compound provided by the invention can identify mitochondria, and detect the fluorescence probe of viscosity；
(2) it can be realized intracellular imaging, study for biology as indicating it in fluorescent biolabels using theoretical basis has been established
Field has potential application；
(3) compound can identify viscosity with high selectivity, will provide new hair for the synthesis of later novel fluorescence probe
Open up direction.
Detailed description of the invention
Fig. 1: EIMV hydrogen nuclear magnetic resonance spectrogram；
Fig. 2: absorption and emission spectrum of the probe in PBS and glycerin solution；
Fig. 3: selectivity of the probe to viscosity.Excitation wavelength is 510 nm；The concentration of probe mother liquor: 10-3M, selective ion
Concentration is 0.4 mM；
Fig. 4: probe fluorescence spectrum in PBS and glycerine different ratio solution；
Excitation wavelength is 510nm；The concentration of probe mother liquor: 10-3M；
Fig. 5: the common location experiment of cell；
Fig. 6: the bio-imaging application of cell viscosity change.
Specific embodiment
Embodiment 1
The synthesis of EIMV:
By 0.32g (2mmol) compound 1,0., 95g (3mmol) compound 2 is dissolved in 20mL methanol, under nitrogen protection
Heating reflux reaction is carried out, overnight, reaction terminates for reaction.By crude product by column chromatography, CH is used2Cl2/CH3The exhibition of OH=70:1
It opens agent progress separating-purifying and obtains compound EIMV.Yield: 45%.1H NMR (400 MHz, DMSO-d 6 ) δ12.85 (s,
1H), 10.58 (s, 1H), 8.80 (d,J=16.0 Hz, 2H), 7.78 (d,J =8.0 Hz, 1H), 7.72
(d,J =8.0 Hz, 1H), 7.55 (t,J =8.0 Hz, 1H), 7.48 (t,J=6.0 Hz, 1H), 7.14
(t,J=8.0 Hz, 1H), 7.06 (d,J=8.0 Hz, 1H), 6.76 (d,J=8.0 Hz, 1H), 4.45
(d,J=6.6 Hz, 2H), 3.34 (s, 1H), 1.78 (s, 6H) 1.43 (t,J=8.0 Hz, 3H).
Embodiment 2
EIMV is in pure aquatic system to the response of viscosity:
Prepare the 10 of 1 part of 10 mL in advance-3Probe M of the inventionN,NThen dimethyl formamide solution takes 12 μ L respectively
It is added in two identical 5mL volumetric flasks, is diluted to 3mL with the solution of PBS in one of bottle, another bottle glycerine
It is diluted to 3mL, then carries out absorbing the detection with emission spectrum, as a result sees Fig. 2.
Conclusion: show that the probe is able to detect viscosity.
Embodiment 3
Selectivity (see Fig. 3) of the EIMV to viscosity:
Wherein: excitation wavelength is 510 nm；The concentration of probe mother liquor: 10-3M.Take 12 μ L probe mother liquors add PBS be diluted to 3ml into
Row, after take the above-mentioned dilution of 3mL, various ions be added carry out spectrum tests and taken wherein various ion concentrations are 0.4mM
12 μ L probe mother liquors add glycerine to be diluted to 3ml progress fluorometric investigation.
Conclusion: it is highly selective that test result shows that probe EIMV has viscosity.
Embodiment 4
Compound EIMV viscosity fluorescence probe with viscosity increase fluorogram variation
Viscosity fluorescence probe prepared by Example 1 is dissolved inN,NIn dimethylformamide, 1mmol/L stock solution is made.From
12 μ L are taken out in stock solution to be added in the centrifuge tube of 3mL, the viscosity number of different proportion is made into PBS and glycerol, are measured
Its photoluminescent property.Fluorescence spectrum is as shown in Fig. 4.
Conclusion: as the increase fluorescence of viscosity gradually increases.
Embodiment 5
Bio-imaging: living cells Coloration experiment
Probe is made into 1mMDMF mother liquor, when dyeing is diluted with 1mL culture medium.
Probe molecule solutions 37oC by inoculated cell in set amount concentration is incubated for 30 min, is washed 3-5 times with PBS；
Then fluorescence imaging is carried out with fluorescence microscope.Excitation wavelength is 560nm, is absorbed in 570-620 nm.
Commercial mitochondrial probe (Mito-tractor) carries out common location experiment, by image under confocal fluorescent microscopic
It knows (Fig. 5), which demonstrates probe can be positioned at mitochondria (see figure 5) well.Common location coefficient is up to 0.9.
Conclusion: illustrate that probe is successfully positioned in mitochondria.
Embodiment 6
Bio-imaging: living cells Coloration experiment
Probe is made into 1 mM DMF mother liquor, when dyeing is diluted with 1mL culture medium.
The identical cell of three groups of incubation times is taken to be tested, one group adds moldin (10um), one group of Jia Monai rhzomorph
(10um), one group of blank, the probe molecule solutions 37oC after being incubated for 30min, then by cell in set amount concentration are incubated for 30 min,
It is washed 3-5 times with PBS, then carries out fluorescence imaging with fluorescence microscope.Excitation wavelength is 560nm, is absorbed in 570-620 nm.
Be added nystatin with not how the cell of rhzomorph (see figure 6) is remarkably reinforced in fluorescence intensity compared to the blank group.
Conclusion: illustrate that probe is able to detect viscosity.
Claims (5)
1. a kind of fluorescence probe for identifying mitochondria viscosity, which is characterized in that the fluorescence probe is named as EIMV, chemistry
Structural formula is as shown in the formula (I):
().
2. the preparation method of fluorescence probe described in claim 1, which comprises the following steps:
Compound 1 and pyridiniujm 2 are made into solvent with methanol, reacts, is heated to reflux under nitrogen protection；Column chromatography obtains chemical combination
Object EIMV；
。
3. application of the fluorescence probe described in claim 1 in terms of spectrum and cell.
4. application of the fluorescence probe described in claim 1 in the measurement of cell mitochondrial viscosity, which is characterized in that described glimmering
Light probe realizes the identification to mitochondria viscosity in a manner of fluorescence enhancement, the measurement applied to cell mitochondrial viscosity.
5. application according to claim 4, which is characterized in that it is glutinous that the fluorescence probe can be applied to living cells mitochondria
Degree measurement.
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Cited By (3)
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| CN112779001A (en) * | 2019-11-02 | 2021-05-11 | 复旦大学 | Preparation and application of near-infrared viscosity fluorescent probe |
| CN113845462A (en) * | 2021-10-26 | 2021-12-28 | 常熟理工学院 | Lysosome fluorescent probe, preparation method and application thereof |
| CN115745969A (en) * | 2022-11-24 | 2023-03-07 | 常熟理工学院 | Fluorescent probe, preparation method and application thereof |
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| CN113845462A (en) * | 2021-10-26 | 2021-12-28 | 常熟理工学院 | Lysosome fluorescent probe, preparation method and application thereof |
| CN115745969A (en) * | 2022-11-24 | 2023-03-07 | 常熟理工学院 | Fluorescent probe, preparation method and application thereof |
| CN115745969B (en) * | 2022-11-24 | 2023-12-29 | 常熟理工学院 | Fluorescent probe, preparation method and application thereof |
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